Joseph R. Nevins

Mechanism of activation of early viral transcription by the adenovirus E1A gene product

Results obtained with certain adenovirus host range mutants (dl312 and hr1) have demonstrated that a functional viral E1A gene is required for the accumulation of early viral mRNA. In this report, it is demonstrated that the failure to accumulate early viral mRNA in E1A mutant-infected cells is due to a lack of transcription of the appropriate transcription units. The E1A gene product appears to enhance transcription of the early transcription units since there is not an absolute requirement for its function. At low multiplicities of infection, transcription from the early transcription units can be detected but at a later time. By increasing the multiplicity of infection, this show activation of early transcription can be enhanced, even though at this multiplicity all input viruses are still mutant. It was also found that early transcription could be detected in mutant-infected cells if the virus was added to cells in which protein synthesis was inhibited prior to infection. Thus inhibition of cellular protein synthesis can mimic the function of the E1A gene. It is proposed that the role of the E1A gene product in the activation of early viral transcription is to inactivate a cellular factor that prevents transcription from the early viral promoters.

Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product

We have attempted to determine whether any cellular genes are activated as a result of the action of the adenoviral E1A gene. The proteins synthesized in uninfected HeLa cells have been compared to those produced in early adenovirus infected cells. At least one protein, absent from uninfected HeLa cells, was synthesized in large amounts following adenovirus infection. This 70 kd protein was not synthesized in cells infected with the E1A mutant dl312, even when the multiplicity of infection with the mutant was such that the only viral gene not expressed was the E1A gene. Thus the induction of the 70 kd protein requires the expression of the viral E1A gene. The 70 kd protein was also induced by heat shock in uninfected cells. The same 70 kd protein is synthesized in 293 cells, a line of human embryonic kidney cells transformed by a fragment of adenovirus DNA. These cells constitutively express the E1A and E1B genes.

The major late adenovirus type-2 transcription unit: Termination is downstream from the last poly(A) site☆

Abstract Late in adenovirus type-2 infection of HeLa cells the majority of viral RNA synthesis occurs from a transcription unit that extends between 16% and 100% on the physical map of the Ad-2 † genome. Poly(A) is added to RNA at five distinct sites within the transcription unit (Fraser & Ziff, 1978; Nevins & Darnell, 1978a,b). As many as 13 different messenger RNAs can be processed from the primary RNA transcripts of this region of the genome (Chow et al., 1977a,b; Nevins & Darnell, 1978a). Examination of nuclear RNA in several different types of experiments (RNA “fingerprint” studies and hybridization of pulse-labeled “nascent” RNA and labeled nuclear RNA synthesized after ultraviolet irradiation to an ordered set of restriction endonuclease-generated DNA fragments) indicates that the mRNA 3′ terminus most distant from the promoter does not correspond to the RNA polymerase termination site for the transcription unit. Transcription, continues ~2500 to 3000 residues beyond the 3′ terminus of the most promoter distal mRNA (localized between co-ordinates 89.7 and 91.9 ) and into the region 98.2–100 on the Ad-2 physical map. The occurrence of RNA termination sites distal to the poly(A) site is discussed as a general feature of transcriptional unit design in animal cells.

The adenovirus-inducible factor E2F stimulates transcription after specific DNA binding.

The promoter-specific factor E2F interacts with critical regulatory sequences within the adenovirus E2 promoter. In addition, the level of active factor increases markedly during a virus infection, dependent on E1A function and coincident with the trans activation of E2 transcription. We have purified the E2F factor through a combination of standard biochemical procedures and DNA affinity chromatography. The purified factor was a single polypeptide of 54,000 molecular weight, as determined by UV crosslinking and renaturation of gel-fractionated protein. Addition of affinity-purified factor to an in vitro transcription system resulted in stimulation of transcription from a promoter containing two E2F-binding sites but not promoters lacking binding sites. We thus conclude that E2F is indeed capable of stimulating transcription once it has bound to the promoter.

N-6-methyl-adenosine in adenovirus type 2 nuclear RNA is conserved in the formation of messenger RNA☆

Abstract In the biogenesis of adenovirus type 2 messenger RNAs, methylation occurs at the 5′ end (cap) and to internal adenosine residues to yield N 6 -methyl-adenosine (m 6 A) (Sommer et al. , 1976; Moss & Koczot, 1976; Wold et al. , 1976). The kinetics of accumulation of 3 H from methyl-labeled methionine and 14 C from uridine into Ad-2 † -specific RNA was measured late in Ad-2 infection. As reported previously (Nevins & Darnell, 1978 a ), the rate of accumulation of [ 14 C]uridine label in nuclear RNA was approximately four- to fivefold faster than in the cytoplasmic RNA, indicating a conservation of about 20% for the total RNA. The initial rates of [ 3 H]methyl label in m 6 A in nuclear RNA and in the cytoplasmic RNA were approximately equal, suggesting a complete (or nearly complete) conservation of m 6 A. In accord with the accumulation kinetics, the ratio of 3 H to 14 C was higher in cytoplasmic RNA than in nuclear RNA that hybridized to equivalent regions of the Ad-2 DNA. A mathematical model was designed to evaluate the accumulation of methyl label in m 6 A, taking into consideration the three major parameters that affect the accumulation curves: equilibration of the S -adenosyl-methionine pool, the nuclear dwell time of sequences destined to be mRNA, and the cytoplasmic stability of mRNA. The half-time ( t 1 2 ) for pool equilibration was determined experimentally to be 22 minutes and the nuclear dwell time and the mean life-time of cytoplasmic mRNA were estimated from 14 C label to be about 30 and 70 minutes, respectively. The model gave an excellent fit to the data when the t 1 2 for pool equilibration time of 24 ± 2 minutes, a nuclear dwell time of 25 ± 10 minutes, and a mean cytoplasmic mRNA life-time of 75 ± 30 minutes were used to evaluate accumulation curves. Even when data from a restricted region of the genome, 40.5–52.6 , which encodes the main portion of at least five 3′ co-terminal mRNAs whose spliced junction with the tripartite leader sequence varies from 38, 40, 43, 45 , and 48 was analyzed, it appeared that m 6 A was conserved. Finally, m 6 A was found to be added in a brief label (3.5 min) mainly to nuclear molecules that were longer than any cytoplasmic RNA. The conservation of m 6 A and its addition prior to splicing raise the possibility that internal methylations are involved, in the formation of mRNA.

Poly(A) site cleavage in a HeLa nuclear extract is dependent on downstream sequences

Efficient utilization of the early SV40 poly(A) site in vivo requires sequences between 5 bp and 18 bp downstream of the cleavage site. We have used a HeLa nuclear extract to examine the sequence requirements for in vitro cleavage. DNA segments containing the SV40 poly(A) site were cloned into an SP6 vector. SP6 RNAs, accurately cleaved and polyadenylated, were detected by primer extension. Cleavage was enhanced by the presence of a cap on the primary transcript, and was inhibited by the addition of 10 microM 7meGpppG. In close agreement with the in vivo results, efficient processing at the poly(A) site in vitro required the specific downstream sequences in the SP6 RNA transcript. These experiments indicate that the sequence in the RNA precursor downstream of the cleavage site, shown to be important for efficient processing in vivo, is recognized in vitro.

Transcription units of adenovirus type 2: Termination of transcription beyond the poly(A) addition site in early regions 2 and 4☆

Abstract Three types of analysis of pulse-labeled nuclear RNA complementary to adenovirus region 2 (~50–75) from cells early in the infectious cycle showed that transcription regularly proceeded past the poly(A) site: (1) nascent chains showed equal molarity of transcription of sequences before and after the poly(A) site at map position 61·6; (2) the size of the nascent RNA molecules containing the sequences distal to the poly(A) site was larger than the size of the molecules proximal to the poly(A) site; (3) ultraviolet light transcription analysis indicated that the sequences beyond the poly(A) site were part of the leftward transcription unit that appears to initiate at map position 75 on the adenovirus type 2 genome. It was also determined that transcription of early region 4 also proceeds beyond the poly(A) addition site (located at map position 91·5), probably terminating in the region of map position 88.

The stability of early adenovirus mRNA is controlled by the viral 72 kd DNA-binding protein

H5ts125, a temperature-sensitive mutant of adenovirus type 5, is restricted to the early phase of infection when grown at the nonpermissive temperature. One phenotype of the virus is the overproduction of early viral mRNA at the nonpermissive temperature relative to levels found in wild-type-infected cells, although normal levels are found at the permissive temperature. We have analyzed this phenomenon for the production of RNA from two specific early viral transcription units, E1A and E1B. Transcription rates from both of these regions were found to be the same in ts125-infected cells as in wild-type-infected cells at the nonpermissive temperature. However, when the cytoplasmic stabilities of the E1A and E1B mRNAs were measured in wild-type- and ts125-infected cells, it was found that at the nonpermissive temperature, the RNAs were 3 to 5 times more stable in a ts125 infection than in a wild-type infection. Since the mutation in ts125 maps to the gene for the 72 kd DNA-binding protein, these results imply that a functional 72 kd protein is required for the rapid turnover of early viral mRNA in wild-type-infected cells, indicating that the abundance of early viral mRNA is controlled by the 72 kd DNA-binding protein.

Processing of late adenovirus nuclear RNA to mRNA. Kinetics of formation of intermediates and demonstration that all events are nuclear.

Abstract Expression of the major late adenovirus transcription unit (which maps between co-ordinates 16 and 99 on the genome) results in the production of five distinct poly(A)-containing RNAs, which are the initial precursors to one of five 3′ coterminal mRNA groups. These initial precursors are processed with a half-life of approximately 30 minutes after the initial poly(A) addition step, which occurs within one to two minutes after transcription of sequences at the poly(A) addition site. The nuclear precursor molecules that eventually yield the fiber mRNA were examined in detail. Four distinct nuclear poly(A)-containing RNAs with 3′ termini at 91·5 were detected. The largest species represents an unspliced molecule and the smallest is the size of the mature mRNA. The two intermediatesized species presumably represent partially spliced molecules. In addition, a precursor product relationship between the species was indicated by kinetic analyses. Finally, it would appear that all major processing steps occur in the nucleus. In the case of the mRNA group that has a 3′ terminus at 51·0, all of the mRNA species of the group were detected in mRNA that had just appeared in the cytoplasm and in approximately the same molar ratios as in samples labeled for longer.

Processing of Adenovirus Nuclear RNA to mRNA

Publisher Summary The mechanism, as well as the regulation, of the biogenesis of adenovirus messenger RNA (mRNA) has been the subject of intensive research over the past several years, principally because it provides an excellent model for the events that occur in the uninfected mammalian cell. The usefulness of adenovirus as a model system is illustrated in this chapter. Adenovirus is a double-stranded DNA virus, which replicates in the nucleus of infected cells. The virus carries no known enzymes into the cell and thus, is completely dependent upon the cellular machinery for at least the initial transcription of its genome. Purified adenovirus DNA can be prepared in large quantities so that defined segments of the genome are available in sufficient amounts for most experiments. The chapter presents a more limited picture of the processes involved in the production of a group of specific mRNAs. As the most comprehensive information available is that dealing with the production of the late adenovirus mRNAs, it is the subject on which the chapter focuses, making reference when appropriate to other systems.